It is well established that transcript elongation by RNA polymerase (RNAP) is a discontinuous process: periods of active RNA synthesis are frequently interrupted by pauses during which RNAP momentarily halts at specific positions along the DNA before resuming normal elongation. Transcriptional pausing by multi-subunit RNAP molecules is remarkably conserved across different organisms, from cancer-associated bacteria to humans, and has been implicated as a key step in a variety of cellular processes. In addition to affecting the overall rate of RNA production, it has been proposed that these pauses facilitate the recruitment of external regulatory factors, the synchronization of transcription wit translation, and the promotion of a variety of RNA processing events, including cotranscriptional folding, splicing, and termination. To date, experiments on transcriptional pausing in bacteria have largely been restricted to in vitro studies. However, it remains to be established whether these pauses persist unchanged in the cellular environment, where both ribosomes and transcription factors may alter transcription dynamics. In this study we aim to bridge the divide between in vitro and in vivo transcription measurements in order to assess the prevalence of pausing across the complete bacterial genome, as well as to determine their role in regulating gene expression. The ability to globally monitor both transcription and translation in vivo was recently pioneered by the Weissman lab. Originally demonstrated in yeast, they showed that RNAP- or ribosome-associated transcripts could be rapidly isolated from live cells, converted into a DNA library without introducing significant bias, and ultimately quantified using massively parallel deep-sequencing techniques. This methodology allows for the identification of transcriptional and translational pause sites across the entire genome with single-nucleotide resolution, and represented a significant advance over other in vivo tracking techniques that suffered from limited spatial and temporal resolution. I aim to further develop high-resolution RNAP profiling by creating a comparable assay capable of monitoring transcription in E. coli. By comparing this transcriptional profiling pause data with previous in vitro studies, I can build a comprehensive top-down model of transcriptional pausing that explains both the molecular mechanism by which RNAP pauses, as well as the function of these pauses in live cells. These maps of RNAP pausing in WT E. coli will also be compared with mutant strains in which RNAP/ribosome coupling is compromised, either through mutations to the ribosome or to transcription factors thought to physically link transcription with translation. The methodologies developed will provide insight into the proliferation of pathogenic bacteria linked with carcinogenesis, such as Helicobacter pylori (H. pylori) and Chlamydia trachomatis (C. trachomatis), as well as providing a useful tool to probe the role transcriptional pausing in human cancer cells.